The separation and purification of principals from complex mixtures routinely employs preparative column chromatography. Column chromatography is a separation technology wherein the mixture to be separated is passed through a column of an adsorbent (the stationary phase) as a solution in a solvent mixture (the mobile phase). A column stationary phase (media) is packed into a bed and is generally porous and has a large surface area through which the liquid mobile phase is pumped. The various compounds of the mixture partition between the stationary phase and the mobile phase according to their relative affinity to the two phases (their partition co-efficient). Since the partition co-efficient is a property unique to the specific chemical structure of each compound in the mixture, the compounds will differentially partition between the phases such that those having higher affinity to the mobile phase will be washed through the column most quickly and those having highest affinity for the stationary phase will be retained in the column longest. This differential of rates of passage through the column thereby accomplishes the desired separation. Complex mixtures of compounds can often be separated with chromatography into single compound pools in a single step procedure. Columns used in chromatography comprise a tubular body enclosing the porous stationary phase through which the mobile phase is pumped with separation effected by the portioning of the components of the mixture to be separated between the mobile and stationary phases. Prior to any separation by chromatography, the packed bed of stationary phase must be prepared starting with a suspension of particles or slurry that has to be introduced into the column. The process of bed formation is called “the packing procedure” and a well packed bed is critical to adequate performance of the separations performed with the chromatographic column. The slurry is uniformly and rapidly compacted into the column under pressure forming the packed bed. The packed bed is maintained under high pressure and density to achieve the most efficient separation of the mixture being chromatographed. The goal of the packing procedure is to provide a bed compressed by the optimum amount of compression.
The porous stationary phase is formed by consolidation of a slurry of discrete particles that is pumped, poured or sucked into the column. Consolidation of the slurry into a packed bed is typically accomplished by filtering it against a particle retaining filter and further compressing the formed filter cake so that it is packed into a volume which is less than the volume that it would have occupied if it had sedimented under the influence of gravity to form a sedimented bed. The efficiency of subsequent chromatographic separations is influenced by the liquid distribution and collection system at the fluid inlet and outlet of the packed bed, but most importantly by the homogeneity of the packed bed formed during packing and its continued stability. If the packed bed is not homogeneous and stable, subsequent chromatographic separations performed with the chromatographic column will be deleteriously effected. Homogeneity and stability of the packed bed depend on the optimum degree of compression which must be determined experimentally for each column diameter, bed height, and bed medium. Several methods are known in the art for packing columns Flow Packing is a method usually used for packing analytical columns (columns of 1 to 10 mm in diameter) and semi-preparative columns (columns of 10 to 100 mm in diameter). In Flow Packing one end of the column is closed by a frit or a filter. The pores of the frit are sized to permit the liquid of the slurry to flow out of the column while preventing discharge of the packing material. At the other end, a slurry of the stationary phase is pumped or poured into the column. A filtration bed builds up against the exit frit and grows until a filter cake is formed. The bed is then compressed further to its target bed height by percolating a number of column volumes (usually more than 5) of a packing solvent at a flow rate that is higher than the flow rates used in operation. Consolidation and subsequent compression take place under the influence of the seepage force, that is the reaction of the bed to the pressure gradient required to maintain flow rate of the packing solvent through the packed bed. Once the bed is compressed by the flow, the solvent flow is stopped, the outlet of the column sealed and an upper end (inlet) cell is adjusted to the target height of the compressed bed. This adjustment is done quickly to avoid a relaxation of the compressed bed exceeding the target bed height. The flow packing method has the disadvantage that beds of packing material compressed in this manner are axially heterogeneous during the flow compression step yielding highest compression close to the column outlet and zero compression at the top of the packed bed. This results in a major relaxation of the bed and a possible re-arrangement of the particles once the packing flow has been stopped and the inlet end has been brought into place. The gradient in bed compression inherent to this method of packing may result in poor bed stability and poor column efficiency depending on the type of medium and the packed bed geometry. This is especially likely in the case of large scale preparative columns.
Flow Packing methods may not be suitable for wide bore columns used in preparative chromatography. Among other factors, it is not desirable to design and fabricate equipment that requires application of a packing flow rate and thus a packing pressure substantially higher than the pressure required for subsequent operation of the large scale of preparative chromatography. To overcome this problem, packing methods which employ mechanical axial compression are used. Axial compression methods achieve the bed compression by an axial movement of a piston slidable within the column, an inlet opening through the piston covered by an inlet frit with pores sized to allow solvent flow but not exit of the packing medium. The need for high liquid pressure in the column body is avoided. A further advantage of the axial compression method is that the bed is compressed homogeneously in an axial direction which avoids the problems of relaxation and particle re-arrangements that occur with the Flow Packing method. Conventionally, when the chromatographic medium within the column is packed, a telescoping rod of a pushing device pushes the piston into the column. This compression packs the packing material to a predetermined pressure. It is the gradient of compression and bed voidage in axial direction that is substantially different between the Flow Packing and the axial compression method.
A disadvantage of axial compression is that columns packed using this method require a means for moving the piston and a means for controlling this movement. Typical methods for the movement are motor drives or hydraulic systems. As these are attached to or built into the column, the cost and mechanical complexity of axial compression columns is substantially greater than for Flow Packing columns. In addition, with the conventional axial compression method of packing chromatographic columns, the pushing device maintains the compression pressure on the packed bed via the piston. This requires that the pushing device remain attached to the column during operation of the chromatographic separation, further complicating the operation of the chromatographic system, especially at preparative scale.
Two contrasting approaches are routinely taken to affect chromatographic separations: 1) the mobile phase is less polar (more organic) than the stationary phase; so called “normal” phase chromatography, and 2) the mobile phase is more polar (aqueous) than the stationary phase; so called “reverse” phase chromatography. Reverse phase chromatography is perceived to have economic advantages over normal phase chromatography due to the usual practice of replacement of the normal phase adsorbent after one or at most a few uses whereas the reverse phase adsorbent can be used for hundreds of separations. The gravitation to reverse phase technology for preparative separations has occurred even in spite of the recognized advantages of normal phase chromatography due to significantly greater capacity per column run and ease of compound recovery from the organic solvent of the mobile phase, both very important advantages for production scale separations. At production scale, the energy required to recycle the normal phase organic solvents is less than that of reversed phase aqueous solvents. The waste disposal costs are reduced for the normal phase organic solvents because of their usually higher BTU content.
Because of the perception that normal phase adsorbents are not reusable or of a very limited useful life, less expensive poor quality normal phase adsorbents are typically employed in preparative column packings. The poorer quality normal phase adsorbent is usually of irregular shaped particles and possesses a wide particle size distribution which together give poor chromatographic performance for the packed bed. High quality normal phase adsorbents are available with spherical particles and narrow particle size distributions. As quality normal phase adsorbent costs about $5,000 per kilogram and is perceived not to be reusable, development of normal phase chromatographic processes has largely been avoided or not considered. With the technologies provided by the present application normal phase chromatographic processes can be developed providing cost savings to users through better performance, higher capacity, easier product recovery, less costly solvent recovery, and less costly solvent disposal.